Membrane RRM2-positive cells represent a malignant population with cancer stem cell features in intrahepatic cholangiocarcinoma

Background Intrahepatic cholangiocarcinoma (iCCA) is one of the most lethal malignancies and highly heterogeneous. We thus aimed to identify and characterize iCCA cell subpopulations with severe malignant features. Methods Transcriptomic datasets from three independent iCCA cohorts (iCCA cohorts 1–3, n = 382) and formalin-fixed and paraffin-embedded tissues from iCCA cohort 4 (n = 31) were used. An unbiased global screening strategy was established, including the transcriptome analysis with the activated malignancy/stemness (MS) signature in iCCA cohorts 1–3 and the mass spectrometry analysis of the sorted stemness reporter-positive iCCA cells. A group of cellular assays and subcutaneous tumor xenograft assay were performed to investigate functional roles of the candidate. Immunohistochemistry was performed in iCCA cohort 4 to examine the expression and localization of the candidate. Molecular and biochemical assays were used to evaluate the membrane localization and functional protein domains of the candidate. Cell sorting was performed and the corresponding cellular molecular assays were utilized to examine cancer stem cell features of the sorted cells. Results The unbiased global screening identified RRM2 as the top candidate, with a significantly higher level in iCCA patients with the MS signature activation and in iCCA cells positive for the stemness reporter. Consistently, silencing RRM2 significantly suppressed iCCA malignancy phenotypes both in vitro and in vivo. Moreover, immunohistochemistry in tumor tissues of iCCA patients revealed an unreported cell membrane localization of RRM2, in contrast to its usual cytoplasmic localization. RRM2 cell membrane localization was then confirmed in iCCA cells via immunofluorescence with or without cell membrane permeabilization, cell fractionation assay and cell surface biotinylation assay. Meanwhile, an unclassical signal peptide and a transmembrane domain of RRM2 were revealed experimentally. They were essential for RRM2 trafficking to cell membrane via the conventional endoplasmic reticulum (ER)–Golgi secretory pathway. Furthermore, the membrane RRM2-positive iCCA cells were successfully sorted. These cells possessed significant cancer stem cell malignant features including cell differentiation ability, self-renewal ability, tumor initiation ability, and stemness/malignancy gene signatures. Patients with membrane RRM2-positive iCCA cells had poor prognosis. Conclusions RRM2 had an alternative cell membrane localization. The membrane RRM2-positive iCCA cells represented a malignant subpopulation with cancer stem cell features. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-024-03174-w.

Expression levels of HCC malignancy and stemness-related genes were determined by RT-qPCR (mean ± SD) in OS4-GFP + and OS4-GFP -Huh7 cells at day 0 after cell sorting.(E) The GFP fluorescence level of GFP + HUCCT1 and GFP -HUCCT1 cells was analyzed by flow cytometry.
Percentage of GFP + cells at day 0 and at the day 5 after cell sorting was quantified.(F) Spheroid formation was performed and compared between OS4-GFP + HUCCT1 cells and OS4-GFP - HUCCT1 cells.Spheroids with diameter ≥ 50μm were counted.(G) OCT4, NANOG, SOX2, and RRM2 levels in OS4-GFP + HUCCT1 cells and OS4-GFP -HUCCT1 cells.Colony formation and cell s 2 migration were performed and compared between OS4-GFP + HUCCT1 cells and OS4-GFP - HUCCT1 cells.(B, C, E, F, G) The Student's t-test was used.

Fig S2. Hierarchical clustering analysis of MS High & MS Low patients and OS4-GFP + & OS4-
GFP -cells.(A-C) Hierarchical clustering analysis of MS High and MS Low patients using genes with significant two-fold higher expression in MS High group vs. MS Low group in each iCCA cohort (146 genes in iCCA cohort 1, 503 genes in iCCA cohort 2, 252 genes in iCCA cohort 3).(D) Heatmap of 301 proteins with significant two-fold higher expression in OS4-GFP + vs. OS4-GFP -RBE cells.
Figure Legends: Fig S1.The effectiveness of MS signature and OS4-GFP reporter in HCC and the implication of OS4-GFP reporter system in HUCCT1 cells.(A)The information of HCC cohorts 1-2 and the hierarchical clustering analysis based on the activation level of MS signature.HCC patients were divided into MS High ，MS Mid and MS Low groups.Hazard ratio of overall survival of MS High group and MS Low group were shown.Log-rank test was performed.(B) Relative expression level of EpCAM and CD24 in MS High group and MS Low group in HCC cohorts 1-2.(C) GFP fluorescence analysis of OS4-GFP + Huh7 and OS4-GFP -Huh7 cells by flow cytometry.Percentage of Huh7 GFP + cells at day 0 or day 3 after cell sorting was quantified.(D)

Fig S3 .
Fig S3.IHC staining scores of RRM2 in iCCA tumors, normal bile duct and hepatocytes.The Student's t-test was performed.Fig S4.The potential transmembrane domain of RRM2 was essential for RRM2 cell membrane localization.(A) The expression of RRM2-Flag, β-catenin and EGFR in RBE and HUCCT1 cells with MG132 and BafA1 treatment.(B) The expression of RRM2 with 36-38aa deletion (ALS del) or 42-44 aa deletion (VLA del) in RBE and HUCCT1 cells.(C) The expression of RRM2 with 223-246aa truncation or mutations of hydrophobic amino acids in 223-246aa region was detected by anti-Flag in HUCCT1 cells.Confocal microscopy images of exogenous RRM2 detected by anti-Flag in HUCCT1 cells.Yellow arrows indicate the cell membrane localization of RRM2.Percentage of RRM2 cell membrane localization was measured (mean ± SD).The Student's t-test was used.

Fig S5 .
Fig S5.RRM2 membrane location by flow cytometry and immunofluorescence.(A) Membrane RRM2 staining in RBE and HUCCT1 cells was determined by flow cytometry using the antibody which recognized 1-111aa of RRM2, with or without GCA treatment.Grey line, no staining; red line, DSMO treatment; blue line, GCA treatment.(B) Confocal microscopy images of endogenous RRM2 and Glogi maker protein GOLPH2, and their co-localization in RBE and HUCCT1 cells.

Fig S6 .
Fig S6.Cell proliferation and DNA replication in RRM2 + iCCA cells and RRM2 -iCCA cells.(A) Cell viability was performed and compared in sorted RRM2 + and RRM2 -cells from both RBE and HUCCT1 iCCA cell lines.Two-way ANOVA analysis was used.(B) Immunofluorescence for EdU labeling in sorted RRM2 + and RRM2 -cells from both RBE and HUCCT1 iCCA cell lines.. Percentage of EdU-positive cells was measured (mean ± SD).The Student's t-test was used.Fig S7.The top 20 enriched signatures in mRRM2 + -like patients in iCCA cohorts 1-3.Red bars represent malignancy-related signatures, orange bars represent progenitor cell-related signatures, dark grey bars represent cell cycle/cell division-related signatures and light grey

Fig S9 .
Fig S9.Overexpression of β-catenin promoted iCCA malignancy features.(A) Overexpression of mutant β-catenin in RBE and HUCCT1 cells was evaluated by western blot.(B-D) colony formation(B), cell migration(C) and spheroid formation(D) were performed in iCCA cells transfected with Ctrl-HA or β-catenin mut -HA.Spheroids with diameter ≥ 50μm were counted.(B, C, D) The Student's t-test was used.Fig S10.Membrane RRM2-positive populations after altering Wnt/β-catenin activation.(A) RBE and HUCCT1 cells were transfected with siCTNNB1 and membrane RRM2 staining was determined by flow cytometry.(B) RBE and HUCCT1 cells were transfected with β-catenin mut and membrane RRM2 staining was determined by flow cytometry.(C) RBE and HUCCT1 cells were treated with LiCl.The protein expression β-catenin was examined with Western blot and membrane RRM2 staining was determined by flow cytometry.Fig S11.The association of RRM2 cell membrane localization and cytoplasm localization.The spearmen correlation of RRM2 cell membrane staining scores and RRM2 cytoplasm staining scores in iCCA cohort 4. Fig S12.The molecular signatures enriched in RRM2 high iCCA group based on the median cut-off of RRM2.(A) iCCAs patients were divided into two subgroups based on the median cutoff of RRM2 (RRM2 high , RRM2 low ).(B) Genes with differential expression between RRM2 high and RRM2 low subgroups.Genes with significant altered expression were used to GSEA analysis.(C) The top 20 enriched signatures in RRM2 high group of iCCA cohorts 1-3.(D) Kaplan-Meier analysis of overall survival for RRM2 High subgroup and RRM2 Low subgroup in iCCA cohorts 1-3.

Fig S13 .
Fig S13.The molecular signatures enriched in RRM2 high iCCA group based on the tertile cut-off of RRM2.(A) The expression of RRM2 in different cohorts.iCCAs patients were divided into three subgroups based on the level of RRM2 (RRM2 high , RRM2 mid , RRM2 low ).(B) Genes with differential expression between RRM2 high and RRM2 low subgroups.Genes with significant altered expression were used to GSEA analysis.(C) The top 20 enriched signatures in RRM2 high group of iCCA cohorts 1-3.(D) Kaplan-Meier analysis of overall survival for RRM2 High subgroup, RRM2 Mid subgroup and RRM2 Low subgroup in iCCA cohorts 1-3.

Fig S1 .Fig
Fig S1.The effectiveness of MS signature and OS4-GFP reporter in HCC and the implication of OS4-GFP reporter system in HUCCT1 cells.

Table S2 . Clinical Characteristics of the MS high and MS low subgroups in iCCA Cohort 1.
a , Fisher's exact test; b , Unpaired t-test; c , Log-rank test s 8

Table S5 . Clinical Characteristics of the RRM2 mem high , mem low , cyto high and cyto low subgroups in iCCA Cohort 4. Clinical Variable Membrane RRM2 staining score Cytosol RRM2 staining score mem high Score ≥ 6 n=14 mem low
a , Fisher's exact test; b , Unpaired t-test; c , Log-rank test